Main interests

Cilia length and motility regulation
Cilia are cellular organelles that protrude from almost every cell membrane. Cilia can be motile or immotile and can appear isolated or in bundles per cell. All types of cilia are thought to have signaling properties. Important signaling pathways during animal development and disease have been related to cilia, such as the Hedgehog, FGF and Notch signaling pathways. When cilia have defective length Hedgehog signaling is abnormal. On the other hand, Notch and FGF signaling defects trigger ciliary length and motility problems. We study cilia length and motility regulation in order to understand ciliopathies. We use the vertebrate zebrafish embryo because it has many ciliated organs with all cilia types and offers excellent imaging and genetic advantages.

Ciliopathies
Cilia have acquired major biomedical relevance due to the disclosure of many different diseases as ciliopathies i.e. diseases that have a ciliary origin. Ciliary protein defects underlie several human syndromes, such as Bardet-Biedl, Alstrom, Joubert, Senior-Løken, Meckel-Gruber, and Oral Facial Digit syndromes, that involve different conjugations of conditions such as polycystic kidney disease, retinal degeneration, anosmia, polydactyly, or organ situs reversal. Several of these syndromes show features not obviously related to cilia, including obesity and mental retardation. Human syndromes such as Kartagener were already identified as ciliopathies affecting motile cilia but many others such as Jeune asphyxiating thoracic dystrophy were not known as ciliopathies and have only recently been allocated to defects in primary cilia signaling. Ciliopathies affect many cell types and organs including kidney, liver, pancreas, heart, lungs, nose, ears, eyes and brain.

Projects

Research on left-right development
This is the main branch of my lab. We have been investigating the role of time in the left-right organizer (LRO). This ciliated embryonic organ is crucial for determining the laterality identity of the vertebrate body-plan. This organ has exciting biophysical properties that control several mechanisms of development, making it an interesting and relevant area of investigation. Namely, biophysical fluid-flow forces that will be translated into gene expression, and determine the location of the future heart on the left and the liver on the right side of the midline axis. Such fluid-flow is directional and known to be generated by motile cilia (Sampaio et al. Dev Cell. 2014). This flow starts by being mild and later becoming strong due to an increase in motile cilia number (Tavares et al. eLIFE. 2017). As a consequence, in the LRO there are two distinct time-windows underlying two fluid-flow regimes. Our current research investigates the molecular mechanisms behind these time-windows by integrating developmental biology, genetics, live-imaging, and bespoke fluid-mechanics modelling in collaboration with the University of Birmingham (Smith et al. Bioarchitecture, 2014; Montenegro-Johnson et al. J Math Biol. 2016; Pintado et al. R. Soc. open sci. 2017).

Research on Primary Ciliary Dyskinesia
The second interest of my lab, still related to left-right development, is the rare disease Primary Ciliary Dyskinesia (PCD), a disease where 50% of the patients develop situs inversus, i.e reversal of the internal organs. Like in most countries, PCD was not properly diagnosed in Portugal. Within the context of COST Beat-PCD-BM1407, my team learned and implemented some of the required techniques according to the European guidelines for PCD. We next triggered the implementation of a Lisbon PCD consortium so that, together with the Hospital Sta Maria (HSM, Lisbon) and Instituto de Medicina Molecular (IMM, Lisbon)), we can currently provide a robust multidisciplinary diagnostic according to the recent requirements for PCD. Our lab performs high-speed videomicroscopy analysis, necessary for the evaluation of cilia beat frequency and pattern (Constant et al. 2018. Acta Pediátrica Portuguesa). As a consequence of PCD diagnostics we started a new area of research in the lab. So far, we have invested on making better diagnostic software for cilia analysis in collaboration with Carla Quintão (NOVA, FCT). Additionally, we have generated zebrafish PCD genetic mutants by using CRISPR-Cas9 gene editing to mimic and better dissect mutations found in patients. The final aim is to rescue some cilia function in patients using gene therapy in collaboration with the Omran lab (WWU, Germany), with whom we have recently been funded by H2020-WIDESPREAD-2016-2017 in the format of a Twinning project, named Lysocil, to improve research on rare diseases in Portugal, including ciliopathies.

Research on Delta-Notch signalling
This signaling pathway is important for ciliary length regulation as well as for deciding the number of motile cilia. We investigated how is DeltaD linked to ciliogenesis and its impact on left-right development (Lopes et al. Development. 2010). This project carried on and we have now identified many interesting genes that are differentially expressed in zebrafish wild-type embryos and deltaD homozygous mutants. Our aim is to understand what are the genes downstream of the Delta-Notch signaling that can rescue cilia length and motility. A recent article from our lab summarizes this branch of our research: ‘Notch/Her12 signalling modulates motile/immotile cilia ratio downstream of Foxj1a in zebrafish left-right organizer’ (Tavares et al. eLIFE. 2017). We will continue searching for Her12 effector genes involved in this fate process.

Research on Pkd2 signalling
This cation channel is thought to partner with Pkd1l1 and sense ‘nodal flow’, an important feature in left-right development. We ask if having no flow has the same impact as having no Pkd2-mediated sensing. We realized this question is much harder to answer then we thought and we are generating new methods to tackle this question. Most interesting, this question led us to another perhaps more fascinating problem which is, how does the embryo try to rescue a defective ‘nodal flow’ during early left-right development?